Activin-actriia antagonists and uses for treating or preventing breast cancer

ABSTRACT

In certain aspects, the present invention provides compositions and methods for treating or preventing breast cancer in humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/899,070, filed Feb. 1, 2007 and U.S. Provisional Application Ser.No. 61/000,540 filed Oct. 25, 2007. All the teachings of theabove-referenced applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Mar. 4, 2010, is namedPHPH018SS.txt, and is 24,551 bytes in size.

BACKGROUND OF THE INVENTION

Breast cancer is the most common type of cancer among women in Westernnations, affecting more than 180,000 women in the U.S. each year. Thedisease arises in the mammary gland, which is made up of a branchingduct system. Each mammary gland, or breast, contains 15 to 20 sectionscalled lobes, and each lobe contains a series of branched ducts thatdrain into the nipple. Epithelial cells that line each duct areresponsible for milk production. Invasive breast cancer is thought tooriginate from normal epithelium of the terminal duct/lobular unitthrough a series of increasingly abnormal proliferative lesions. Oncethe tumor acquires the ability to metastasize, breast cancer cellsspread to other organs, making treatment increasingly more difficult.The most common sites of breast cancer metastases are the lung, liver,and bones. Metastases to the bone are commonly associated with severepain, loss of bone, and increased risk of fractures. Manyanti-estrogenic therapies used in the treatment of breast cancer arealso associated with accelerated bone loss.

Patients diagnosed with breast cancer typically undergo surgery and/orradiotherapy to treat the primary tumor, followed by adjuvant therapy totreat any cancer cells that may have spread to distant sites. Adjuvanttherapy consists of cytotoxic chemotherapy and/or endocrine therapy.Although chemotherapy has been effective in treating various types ofmalignancies, many anti-neoplastic compounds induce undesirable sideeffects. Additionally, many tumors either fail to respond or becomeresistant to chemotherapy and endocrine therapies. While adjuvanttherapy has improved the mortality rate among breast cancer patients,the 10-year survival rate for patients with the most commonhistopathological types of invasive breast cancer is still only 35-50%(Weigelt et al. 2005 Nat. Rev. Cancer 5: 591-602). Further, due to poorprognosis criteria, many women that would be cured by local treatmentalone receive adjuvant therapy unnecessarily.

Consequently, more efficient and effective molecular targets againstbreast cancer are needed. Alternative therapies that are less toxicand/or more effective than chemotherapy and endocrine therapies wouldimprove treatment regimens and increase survival. Further, agents thatcan be used as preventative treatments for patients that may be at riskfor developing invasive or metastatic breast cancer would be useful inthe clinic. It is an object of the present disclosure, therefore, toprovide alternative compositions and methods for treating breast canceror inhibiting or preventing the progression of breast cancer inpatients.

SUMMARY OF THE INVENTION

In part, the disclosure relates to the use of activin antagonists, aswell as ActRIIa antagonists, to treat or prevent breast cancer or boneloss associated with breast cancer. In particular, the disclosureprovides methods for treating or preventing breast cancer using asoluble form of ActRIIa that acts as an inhibitor of activin. Whilesoluble ActRIIa may affect cancer cell growth or survival through amechanism other than activin antagonism, desirable therapeutic agentsmay nonetheless be selected on the basis of activin antagonism orActRIIa antagonism or both. Such agents are referred to collectively asactivin-ActRIIa antagonists. Therefore, in certain embodiments, thedisclosure provides methods for using activin-ActRIIa antagonists,including, for example, activin-binding ActRIIa polypeptides,anti-activin antibodies, anti-ActRIIa antibodies, activin- orActRIIa-targeted small molecules and aptamers, and nucleic acids thatdecrease expression of activin and ActRIIa, to treat or prevent breastcancer in patients in need thereof. As described in U.S. patentapplication Ser. No. 11/603,485, incorporated by reference herein,activin-ActRIIa antagonists can be used to promote bone growth andincrease bone density. As described herein, such antagonists can also beused to treat or prevent breast cancer, breast cancer metastases to thebone and bone loss associated with breast cancer.

In certain aspects, the disclosure provides methods for treating orpreventing breast cancer using polypeptides comprising a soluble,activin-binding ActRIIa polypeptide that binds to activin. ActRIIapolypeptides may be formulated as a pharmaceutical preparationcomprising the activin-binding ActRIIa polypeptide and apharmaceutically acceptable carrier. The activin-binding ActRIIapolypeptide may bind to activin with a K_(D) less than 1 micromolar orless than 100, 10 or 1 nanomolar. Optionally, the activin-bindingActRIIa polypeptide selectively binds activin versus GDF11 and/or GDF8,and optionally with a K_(D) that is at least 10-fold, 20-fold or 50-foldlower with respect to activin than with respect to GDF11 and/or GDF8.While not wishing to be bound to a particular mechanism of action, it isexpected that this degree of selectivity for activin inhibition overGDF11/GDF8 inhibition accounts for effects on bone or tumor survival orgrowth without a consistently measurable effect on muscle. In manyembodiments, an ActRIIa polypeptide will be selected for causing lessthan 15%, less than 10% or less than 5% increase in muscle at doses thatachieve desirable effects on cancer cells. The composition may be atleast 95% pure, with respect to other polypeptide components, asassessed by size exclusion chromatography, and optionally, thecomposition is at least 98% pure. An activin-binding ActRIIa polypeptidefor use in such a preparation may be any of those disclosed herein, suchas a polypeptide having an amino acid sequence selected from SEQ ID NOs:2, 3, 7 or 12, or having an amino acid sequence that is at least 80%,85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selectedfrom SEQ ID NOs: 2, 3, 7, 12 or 13. An activin-binding ActRIIapolypeptide may include a functional fragment of a natural ActRIIapolypeptide, such as one comprising at least 10, 20, 30, 50 or 90 ormore amino acids of a sequence selected from SEQ ID NOs: 1-3 or asequence of SEQ ID NO: 2, lacking the C-terminal 10 to 15 amino acids(the “tail”).

A soluble, activin-binding ActRIIa polypeptide may include one or morealterations in the amino acid sequence (e.g., in the ligand-bindingdomain) relative to a naturally occurring ActRIIa polypeptide. Examplesof altered ActRIIa polypeptides are provided in WO 2006/012627, pp.59-60, incorporated by reference herein. The alteration in the aminoacid sequence may, for example, alter glycosylation of the polypeptidewhen produced in a mammalian, insect or other eukaryotic cell or alterproteolytic cleavage of the polypeptide relative to the naturallyoccurring ActRIIa polypeptide.

An activin-binding ActRIIa polypeptide may be a fusion protein that has,as one domain, an ActRIIa polypeptide (e.g., a ligand-binding portion ofan ActRIIa) and one or more additional domains that provide a desirableproperty, such as improved pharmacokinetics, easier purification,enhanced targeting to particular tissues, etc. For example, a domain ofa fusion protein may enhance one or more of in vivo stability, in vivohalf life, uptake/administration, tissue localization or distribution,formation of protein complexes, multimerization of the fusion protein,and/or purification. An activin-binding ActRIIa fusion protein mayinclude an immunoglobulin Fc domain (wild-type or mutant) or a serumalbumin or other polypeptide portion that provides desirable propertiessuch as improved pharmacokinetics, improved solubility or improvedstability. In a preferred embodiment, an ActRIIa-Fc fusion comprises arelatively unstructured linker positioned between the Fc domain and theextracellular ActRIIa domain. This unstructured linker may correspond tothe roughly 15 amino acid unstructured region at the C-terminal end ofthe extracellular domain of ActRIIa (the “tail”), or it may be anartificial sequence of 1, 2, 3, 4 or 5 amino acids or a length ofbetween 5 and 15, 20, 30, 50 or more amino acids that are relativelyfree of secondary structure, or a mixture of both. A linker may be richin glycine and proline residues and may, for example, contain a singlesequence of threonine/serine and glycines or repeating sequences ofthreonine/serine and glycines (e.g., TG₄ (SEQ ID NO: 15) or SG₄ (SEQ IDNO: 16) singlets or repeats). A fusion protein may include apurification subsequence, such as an epitope tag, a FLAG tag, apolyhistidine sequence, and a GST fusion. Optionally, a soluble ActRIIapolypeptide includes one or more modified amino acid residues selectedfrom: a glycosylated amino acid, a PEGylated amino acid, a farnesylatedamino acid, an acetylated amino acid, a biotinylated amino acid, anamino acid conjugated to a lipid moiety, and an amino acid conjugated toan organic derivatizing agent. A pharmaceutical preparation may alsoinclude one or more additional compounds such as a compound that is usedto treat a bone disorder. Preferably, a pharmaceutical preparation issubstantially pyrogen free. In general, it is preferable that an ActRIIaprotein be expressed in a mammalian cell line that mediates suitablynatural glycosylation of the ActRIIa protein so as to diminish thelikelihood of an unfavorable immune response in a patient. Human and CHOcell lines have been used successfully, and it is expected that othercommon mammalian expression systems will be useful. Additionally, yeastand other cell types have been genetically altered to express mammalianenzymes that catalyze glycosylation, thus allowing the generation oftightly controlled mammalian-like glycosylation on proteins expressed inthese non-mammalian cells. These recombinant cell lines may also be usedto express the proteins described herein.

As described herein, ActRIIa proteins designated ActRIIa-Fc (a form witha minimal linker between the ActRIIa portion and the Fc portion) havedesirable properties, including selective binding to activin versus GDF8and/or GDF11, high affinity ligand binding and serum half life greaterthan two weeks in animal models. In certain embodiments the inventionprovides methods for treating or preventing breast cancer usingActRIIa-Fc polypeptides and pharmaceutical preparations comprising suchpolypeptides and a pharmaceutically acceptable excipient.

In certain aspects, the disclosure provides methods for treating orpreventing breast cancer using nucleic acids encoding a solubleactivin-binding ActRIIa polypeptide. An isolated polynucleotide maycomprise a coding sequence for a soluble, activin-binding ActRIIapolypeptide, such as described above. For example, an isolated nucleicacid may include a sequence coding for an extracellular domain (e.g.,ligand-binding domain) of an ActRIIa and a sequence that would code forpart or all of the transmembrane domain and/or the cytoplasmic domain ofan ActRIIa, but for a stop codon positioned within the transmembranedomain or the cytoplasmic domain, or positioned between theextracellular domain and the transmembrane domain or cytoplasmic domain.For example, an isolated polynucleotide may comprise a full-lengthActRIIa polynucleotide sequence such as SEQ ID NO: 4 or 5, or apartially truncated version, said isolated polynucleotide furthercomprising a transcription termination codon at least six hundrednucleotides before the 3′-terminus or otherwise positioned such thattranslation of the polynucleotide gives rise to an extracellular domainoptionally fused to a truncated portion of a full-length ActRIIa. Apreferred nucleic acid sequence is SEQ ID NO: 14. Nucleic acids usefulin accordance with the methods described herein may be operably linkedto a promoter for expression, and the disclosure provides cellstransformed with such recombinant polynucleotides. Preferably the cellis a mammalian cell such as a Chinese hamster ovary (CHO) cell.

The disclosure also provides methods for making a soluble,activin-binding ActRIIa polypeptide that can be used for treating orpreventing breast cancer. Such a method may include expressing any ofthe nucleic acids (e.g., SEQ ID NO: 4, 5 or 14) disclosed herein in asuitable cell, such as a CHO cell. Such a method may comprise: a)culturing a cell under conditions suitable for expression of the solubleActRIIa polypeptide, wherein said cell is transformed with a solubleActRIIa expression construct; and b) recovering the soluble ActRIIapolypeptide so expressed. Soluble ActRIIa polypeptides may be recoveredas crude, partially purified or highly purified fractions. Purificationmay be achieved by a series of purification steps, including, forexample, one, two or three or more of the following, in any order:protein A chromatography, anion exchange chromatography (e.g., Qsepharose), hydrophobic interaction chromatography (e.g.,phenylsepharose), size exclusion chromatography, and cation exchangechromatography.

In certain aspects, an activin-ActRIIa antagonist disclosed herein, suchas a soluble, activin-binding ActRIIa polypeptide, may be used in amethod for treating, preventing or inhibiting breast cancer in asubject, including, for example, methods for delaying the onset ofbreast cancer, inhibiting the progression of breast cancer, reducingtumor size, preventing tumor growth, delaying the onset of metastasis orpreventing metastasis, including metastasis to bone. In certainembodiments, the disclosure provides methods for decreasing orinhibiting the growth or survival of breast cancer cells in patients inneed thereof. A method may comprise administering to a subject in needthereof an effective amount of activin-ActRIIa antagonist. In certainaspects, the disclosure provides uses of activin-ActRIIa antagonists formaking a medicament for the treatment or prevention of breast cancer asdescribed herein. The disclosure also relates to combination therapiescomprising an activin-ActRIIa antagonist and radiation therapy,chemotherapy (e.g., a cytotoxic agent), and/or endocrine therapy. Theantagonist may be an ActRIIa-Fc fusion protein, wherein the ActRIIa-Fcfusion protein comprises an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO:3.

In further embodiments, the present invention relates to methods ofpreventing or delaying the onset of breast cancer in patients with oneor more breast cancer risk factors. In some embodiments, the inventionrelates to methods of preventing or delaying the onset of metastaticdisease in patients already diagnosed with a primary breast tumor orwith a proliferative lesion of the breast. The method of preventing ordelaying the onset of breast cancer in a human patient may compriseadministering to a human patient in need thereof an effective amount ofa polypeptide selected from the group consisting of: a) a polypeptidecomprising an amino acid sequence at least 90% identical to SEQ ID NO:2;b) a polypeptide comprising an amino acid sequence at least 90%identical to SEQ ID NO:3; and c) a polypeptide comprising at least 50consecutive amino acids selected from SEQ ID NO: 2.

Other embodiments of the invention relate to a method of inhibitingactivin-mediated signaling in a human patient with breast cancer. Incertain embodiments, the method comprises administering to the humanpatient an effective amount of an activin-ActRIIa antagonist. In furtherembodiments, the antagonist is a polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence atleast 90% identical to SEQ ID NO:2; b) a polypeptide comprising an aminoacid sequence at least 90% identical to SEQ ID NO:3; and c) apolypeptide comprising at least 50 consecutive amino acids selected fromSEQ ID NO: 2.

In certain aspects, the disclosure provides a method for identifying anagent that inhibits the growth or survival of cancer cells (e.g., breastcancer cells). The method comprises: a) identifying a test agent thatbinds to activin or a ligand-binding domain of an ActRIIa polypeptide;and b) evaluating the effect of the agent on the proliferation,survival, or apoptosis of cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the purification of ActRIIa-hFc expressed in CHO cells. Theprotein purifies as a single, well-defined peak as visualized by sizingcolumn (left panel) and Coomassie stained SDS-PAGE (right panel) (leftlane: molecular weight standards; right lane: ActRIIa-hFc).

FIG. 2 shows the binding of ActRIIa-hFc to activin and GDF-11, asmeasured by BiaCore™ assay.

FIG. 3 shows that ActRIIa-mFc treatment greatly reduces the formation ofmetastatic lesions in a mouse model of metastatic breast cancer. Micewere visualized non-invasively (anaesthetized, fluorescent imaging) fiveweeks after intracardiac injection of luciferase expressing MDA-MB-231breast cancer cells. 12/14 vehicle treated mice show visible metastaticlesions, while only 4/12 ActRIIa-mFc treated mice show visible lesions.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general branches: the BMP/GDF and the TGF-beta/Activinbranches, whose members have diverse, often complementary effects. Bymanipulating the activity of a member of the TGF-beta family, it isoften possible to cause significant physiological changes, in anorganism. For example, the Piedmontese and Belgian Blue cattle breedscarry a loss-of-function mutation in the GDF8 (also called myostatin)gene that causes a marked increase in muscle mass. Grobet et al., Nat.Genet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles ofGDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Activins are dimeric polypeptide growth factors that belong to theTGF-beta superfamily. There are three principal activin forms (A, B, andAB) that are homo/heterodimers of two closely related β subunits(β_(A)β_(A), β_(B)β_(B), and β_(A)β_(B), respectively). The human genomealso encodes an activin C and an activin E, which are primarilyexpressed in the liver, and heterodimeric forms containing β_(C) orβ_(E) are also known. In the TGF-beta superfamily, activins are uniqueand multifunctional factors that can stimulate hormone production inovarian and placental cells, support neuronal cell survival, influencecell-cycle progress positively or negatively depending on cell type, andinduce mesodermal differentiation at least in amphibian embryos (DePaoloet al., 1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997,Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963). Inaddition, it has been shown that activin B is involved in the regulationof mammary epithelial cell differentiation in mice (Robinson andHennighausen, 1997 Development 124: 2701-2708). In several tissues,activin signaling is antagonized by its related heterodimer, inhibin.For example, during the release of follicle-stimulating hormone (FSH)from the pituitary, activin promotes FSH secretion and synthesis, whileinhibin prevents FSH secretion and synthesis. Other proteins that mayregulate activin bioactivity and/or bind to activin include follistatin(FS), follistatin-related protein (FSRP) and α₂-macroglobulin.

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat.Rev. Mol. Cell. Biol. 1:169-178). These type I and type II receptors aretransmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling; and type II receptors arerequired for binding ligands and for expression of type I receptors.Type I and II activin receptors form a stable complex after ligandbinding, resulting in phosphorylation of type I receptors by type IIreceptors.

Two related type II receptors, ActRIIa and ActRIIb, have been identifiedas the type II receptors for activins (Mathews and Vale, 1991, Cell65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins,ActRIIa and ActRIIb can biochemically interact with several other TGF-βfamily proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita etal., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc.Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell. 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is the primarytype I receptor for activins, particularly for activin A, and ALK-7 mayserve as a receptor for activins as well, particularly for activin B.

As described herein, a soluble ActRIIa polypeptide (sActRlIa), whichshows substantial preference in binding to activin A as opposed to otherTGF-beta family members, such as GDF8 or GDF11, may be used to treat orprevent cancer, particularly breast cancer. While not wishing to bebound to any particular mechanism, it is expected that the effect ofsActRIIa is caused primarily by an activin antagonist effect, given thevery strong activin binding (picomolar dissociation constant) exhibitedby the particular sActRIIa construct used in these studies.Activin-ActRIIa antagonists include, for example, activin-bindingsoluble ActRIIa polypeptides, antibodies that bind to activin(particularly the activin A or B subunits, also referred to as βA or βB)and disrupt ActRIIa binding, antibodies that bind to ActRIIa and disruptactivin binding, non-antibody proteins selected for activin or ActRIIabinding (see e.g., WO/2002/088171, WO/2006/055689, and WO/2002/032925for examples of such proteins and methods for design and selection ofsame), randomized peptides selected for activin or ActRIIa binding,often affixed to an Fc domain. Two different proteins (or othermoieties) with activin or ActRIIa binding activity, especially activinbinders that block the type I (e.g., a soluble type I activin receptor)and type II (e.g., a soluble type II activin receptor) binding sites,respectively, may be linked together to create a bifunctional bindingmolecule. Nucleic acid aptamers, small molecules and other agents thatinhibit the activin-ActRIIa signaling axis may also be used. Variousproteins have activin-ActRIIa antagonist activity, including inhibin(i.e., inhibin alpha subunit), although inhibin does not universallyantagonize activin in all tissues, follistatin (e.g., follistatin-288and follistatin-315), FSRP, activin C, alpha(2)-macroglobulin, and anM108A (methionine to alanine change at position 108) mutant activin A.Generally, alternative forms of activin, particularly those withalterations in the type I receptor binding domain can bind to type IIreceptors and fail to form an active ternary complex, thus acting asantagonists. Additionally, nucleic acids, such as antisense molecules,siRNAs or ribozymes that inhibit activin A, B, C or E, or, particularly,ActRIIa expression, can be used as activin-ActRIIa antagonists. Theactivin-ActRIIa antagonist to be used may exhibit selectivity forinhibiting activin-mediated signaling versus other members of theTGF-beta family, and particularly with respect to GDF8 and GDF11.Soluble ActRIIb proteins do bind to activin, however, the wild typeprotein does not exhibit significant selectivity in binding to activinversus GDF8/11. Nonetheless, such ActRIIb polypeptides, as well asaltered forms of ActRIIb with different binding properties (see, e.g.,WO 2006/012627, pp. 55-59, incorporated herein by reference) may achievethe desired effects on cancer cells. Native or altered ActRIIb may begiven added specificity for activin by coupling with a second,activin-selective binding agent.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

The term “breast cancer” refers to any proliferative lesion orproliferative abnormality of the breast including, for example, benignlesions, pre-malignant and malignant lesions, solid tumors, andmetastatic disease (both locally metastatic, e.g., stage III, and morewidely metastatic, e.g., stage 1V). Breast cancer includes but is notlimited to adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer. Breast cancer also refers to disease inother organs such as lung, liver, and bone, that originated from ametastatic lesion in the breast. Breast cancer also encompasses bothhormone-responsive and hormone-independent cancers. Generally,hormone-independent breast cancers are characterized by the absence orreduced levels of estrogen and/or progesterone receptors, and thesecancers are typically refractory to treatment with antihormonal(especially antiestrogenic) therapies. Breast cancers are alsocategorized on the basis of Her2 expression, with Her2⁺ tumors having aworse prognosis than Her2⁻ tumors.

2. ActRIIa Polypeptides

In certain aspects, the present invention relates to methods fortreating or preventing breast cancer using ActRIIa polypeptides. As usedherein, the term “ActRIIa” refers to a family of activin receptor typeIIa (ActRIIa) proteins from any species and variants derived from suchActRIIa proteins by mutagenesis or other modification. Reference toActRIIa herein is understood to be a reference to any one of thecurrently identified forms. Members of the ActRIIa family are generallytransmembrane proteins, composed of a ligand-binding extracellulardomain with a cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine kinase activity.

The term “ActRIIa polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIa family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. See, for example,WO/2006/012627. For example, ActRIIa polypeptides include polypeptidesderived from the sequence of any known ActRIIa having a sequence atleast about 80% identical to the sequence of an ActRIIa polypeptide, andoptionally at least 85%, 90%, 95%, 97%, 99% or greater identity. Forexample, an ActRIIa polypeptide of the invention may bind to and inhibitthe function of an ActRIIa protein and/or activin. An ActRIIapolypeptide may be selected for activity in inhibiting cancer cellproliferation or survival in vivo. Examples of ActRIIa polypeptidesinclude human ActRIIa precursor polypeptide (SEQ ID NO: 1) and solublehuman ActRIIa polypeptides (e.g., SEQ ID NOs: 2, 3, 7 and 12).

The human ActRIIa precursor protein sequence is as follows:

(SEQ ID NO: 1) MGAAAKLAFAVFLISCSSGA ILGRSETQECLFFNANWEKDRT N QTGVEPCYGDKDKRRHCFATWK N ISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTM VTNVDFPPKESSL

The signal peptide is single underlined; the extracellular domain is inbold and the potential N-linked glycosylation sites are doubleunderlined.

The human ActRIIa soluble (extracellular), processed polypeptidesequence is as follows:

(SEQ ID NO: 2) ILGRSETQECLFFNANWEKLRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSNPVTPKPP

It should be noted that the N-terminal sequence beginning “ILG . . . ”has been experimentally determined and differs from the “AIL . . . ”N-terminal sequence that is commonly proposed in the literature. TheC-temminal “tail” of the extracellular domain is underlined. Thesequence with the “tail” deleted (a A 15 sequence) is as follows:

(SEQ ID NO: 3) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFP EM

The nucleic acid sequence encoding human ActRIIa precursor protein is asfollows (nucleotides 164-1705 of Genbank entry NM001616):

(SEQ ID NO: 4) ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATCTCCTGTTCTTCAGGTGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCTATTACAACATCCTGCTCTATTCCTTGGTGCCACTTATGTTAATTGCGGGGATTGTCATTTGTGCATTTTGGGTGTACAGGCATCACAAGATGGCCTACCCTCCTGTACTTGTTCCAACTCAAGACCCAGGACCACCCCCACCTTCTCCATTACTAGGGTTGAAACCACTGCAGTTATTAGAAGTGAAAGCAAGGGGAAGATTTGGTTGTGTCTGGAAAGCCCAGTTGCTTAACGAATATGTGGCTGTCAAAATATTTCCAATACAGGACAAACAGTCATGGCAAAATGAATACGAAGTCTACAGTTTGCCTGGAATGAAGCATGAGAACATATTACAGTTCATTGGTGCAGAAAAACGAGGCACCAGTGTTGATGTGGATCTTTGGCTGATGACAGCATTTCATGAAAAGGGTTCACTATCAGACTTTCTTAAGGCTAATGTGGTCTCTTGGAATGAACTGTGTCATATTGCAGAAACCATGGCTAGAGGATTGGCATATTTACATGAGGATATACCTGGCCTAAAAGATGGCCACAAACCTGCCATATCTCACAGGGACATCAAAAGTAAAAATGTGCTGTTGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATTTGAGGCTGGCAAGTCTGCAGGCGATACCCATGGACAGGTTGGTACCCGGAGGTACATGGCTCCAGAGGTATTAGAGGGTGCTATAAACTTCCAAAGGGATGCATTTTTGAGGATAGATATGTATGCCATGGGATTAGTCCTATGGGAACTGGCTTCTCGCTGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTGCCATTTGAGGAGGAAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTGTGCATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAACATGCTGGAATGGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGACGCAGAAGCCAGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATGCAGAGACTAACAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAATGGTGACAAATGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA

The nucleic acid sequence encoding a human ActRIIa soluble(extracellular) polypeptide is as follows:

(SEQ ID NO: 5) ATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCC

In a specific embodiment, the invention relates to methods for treatingor preventing breast cancer using soluble ActRIIa polypeptides. Asdescribed herein, the term “soluble ActRIIa polypeptide” generallyrefers to polypeptides comprising an extracellular domain of an ActRIIaprotein. The term “soluble ActRIIa polypeptide,” as used herein,includes any naturally occurring extracellular domain of an ActRIIaprotein as well as any variants thereof (including mutants, fragmentsand peptidomimetic forms). An activin-binding ActRIIa polypeptide is onethat retains the ability to bind to activin, including, for example,activin AA, AB, BB, or forms that include a C or E subunit. Optionally,an activin-binding ActRIIa polypeptide will bind to activin AA with adissociation constant of 1 nM or less. The extracellular domain of anActRIIa protein binds to activin and is generally soluble, and thus canbe termed a soluble, activin-binding ActRIIa polypeptide. Examples ofsoluble, activin-binding ActRIIa polypeptides include the solublepolypeptide illustrated in SEQ ID NOs: 2, 3, 7, 12 and 13. SEQ ID NO:7is referred to as ActRIIa-hFc, and is described further in the Examples.Other examples of soluble, activin-binding ActRIIa polypeptides comprisea signal sequence in addition to the extracellular domain of an ActRIIaprotein, for example, the honey bee melitin leader sequence (SEQ ID NO:8), the tissue plaminogen activator (TPA) leader (SEQ ID NO: 9) or thenative ActRIIa leader (SEQ ID NO: 10). The ActRIIa-hFc polypeptideillustrated in SEQ ID NO: 13 uses a tPA leader.

Functionally active fragments of ActRIIa polypeptides can be obtained byscreening polypeptides recombinantly produced from the correspondingfragment of the nucleic acid encoding an ActRIIa polypeptide. Inaddition, fragments can be chemically synthesized using techniques knownin the art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. The fragments can be produced (recombinantly or by chemicalsynthesis) and tested to identify those peptidyl fragments that canfunction as antagonists (inhibitors) of ActRIIa protein or signalingmediated by activin.

Functionally active variants of ActRIIa polypeptides can be obtained byscreening libraries of modified polypeptides recombinantly produced fromthe corresponding mutagenized nucleic acids encoding an ActRIIapolypeptide. The variants can be produced and tested to identify thosethat can function as antagonists (inhibitors) of ActRIIa protein orsignaling mediated, by activin. In certain embodiments, a functionalvariant of the ActRIIa polypeptides comprises an amino acid sequencethat is at least 75% identical to an amino acid sequence selected fromSEQ ID NOs: 2 or 3. In certain cases, the functional variant has anamino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3.

Functional variants may be generated by modifying the structure of anActRIIa polypeptide for such purposes as enhancing therapeutic efficacy,or stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo). Such modified ActRIIa polypeptides when selectedto retain activin binding, are considered functional equivalents of thenaturally-occurring ActRIIa polypeptides. Modified ActRIIa polypeptidescan also be produced, for instance, by amino acid substitution,deletion, or addition. For instance, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(e.g., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Whether a change in the amino acidsequence of an ActRIIa polypeptide results in a functional homolog canbe readily determined by assessing the ability of the variant ActRIIapolypeptide to produce a response in cells in a fashion similar to thewild-type ActRIIa polypeptide.

In certain embodiments, the present invention contemplates methods fortreating or preventing breast cancer using ActRIIa polypeptides havingspecific mutations that alter the glycosylation of the polypeptide. Suchmutations may be selected so as to introduce or eliminate one or moreglycosylation sites, such as O-linked or N-linked glycosylation sites.Asparagine-linked glycosylation recognition sites generally comprise atripeptide sequence, asparagine-X-threonine or asparagine-X-serine(where “X” is any amino acid) which is specifically recognized byappropriate cellular glycosylation enzymes. The alteration may also bemade by the addition of, or substitution by, one or more serine orthreonine residues to the sequence of the wild-type ActRIIa polypeptide(for O-linked glycosylation sites). A variety of amino acidsubstitutions or deletions at one or both of the first or third aminoacid positions of a glycosylation recognition site (and/or amino aciddeletion at the second position) results in non-glycosylation at themodified tripeptide sequence. Another means of increasing the number ofcarbohydrate moieties on an ActRIIa polypeptide is by chemical orenzymatic coupling of glycosides to the ActRIIa polypeptide. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine; (b) free carboxyl groups; (c) free sulfhydryl groups suchas those of cysteine; (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (f) the amide group ofglutamine. Removal of one or more carbohydrate moieties present on anActRIIa polypeptide may be accomplished chemically and/or enzymatically.Chemical deglycosylation may involve, for example, exposure of theActRIIa polypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the amino acid sequence intact.Enzymatic cleavage of carbohydrate moieties on ActRIIa polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al. (1987) Meth. Enzymol. 138:350. Thesequence of an ActRIIa polypeptide may be adjusted, as appropriate,depending on the type of expression system used, as mammalian, yeast,insect and plant cells may all introduce differing glycosylationpatterns that can be affected by the amino acid sequence of the peptide.In general, ActRIIa proteins for use in humans may be expressed in amammalian cell line that provides proper glycosylation, such as HEK293or CHO cell lines, although other mammalian expression cell lines areexpected to be useful as well.

This disclosure further contemplates methods of generating mutants,particularly sets of combinatorial mutants of an ActRIIa polypeptide, aswell as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, ActRIIa polypeptide variants which bind to activin or otherligands. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ActRIIapolypeptide variant may be screened for ability to bind to an ActRIIaligand, to prevent binding of an ActRIIa ligand to an ActRIIapolypeptide or to interfere with signaling caused by an ActRIIa ligand.

The activity of an ActRIIa polypeptide or its variants may also betested in a cell-based or in vivo assay. For example, the effect of anActRIIa polypeptide variant on the proliferation or survival of cancercells may be assessed. Cancer cells may refer to cells in a livingsubject that make up a solid tumor or to cells that have originated froma tumor and that have spread to other sites within a living subject(i.e., metastatic cells). Additionally, cancer cells may refer to cellsobtained or derived from a tumor or cancerous growth and that arecultured in vitro. Cancer cells also encompass cell lines that may becultivated in vitro or used in animal xenograft studies, for example.Cancer cells also refer to cells derived from metastatic cells throughcell division following metastasis. The cells may be hormone-responsive(e.g., estrogen receptor positive) or hormone-independent (e.g.,estrogen receptor negative). Cancer cell proliferation or survival maybe assessed in the presence of one or more recombinant ActRIIa ligandproteins (e.g., activin), and cells may be transfected so as to producean ActRIIa polypeptide and/or variants thereof, and optionally, anActRIIa ligand. Likewise, an ActRIIa polypeptide may be administered toa mouse or other animal, and one or more measurements, such as tumorsize, or the rate of cell proliferation or apoptosis relative to acontrol, may be assessed.

Combinatorially-derived variants can be generated which have a selectiveor generally increased potency relative to a naturally occurring ActRIIapolypeptide. Likewise, mutagenesis can give rise to variants which haveintracellular half-lives dramatically different than the correspondingwild-type ActRIIa polypeptide. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular processes which result in destruction of, or otherwiseinactivation of a native ActRIIa polypeptide. Such variants, and thegenes which encode them, can be utilized to alter ActRIIa polypeptidelevels by modulating the half-life of the ActRIIa polypeptides. Forinstance, a short half-life can give rise to more transient biologicaleffects and, when part of an inducible expression system, can allowtighter control of recombinant ActRIIa polypeptide levels within thecell. In an Fc fusion protein, mutations may be made in the linker (ifany) and/or the Fc portion to alter the half-life of the protein.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ActRIIa polypeptide sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ActRIIapolypeptide nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes can then be ligated into anappropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevierpp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakuraet al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res.11:477). Such techniques have been employed in the directed evolution ofother proteins (see, for example, Scott et al., (1990) Science249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin etal., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ActRIIa polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell. Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of ActRIIa polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ActRIIa polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include activin binding assays and activin-mediated cellsignaling assays.

In certain embodiments, ActRIIa polypeptides useful in accordance withthe methods described herein may further comprise post-translationalmodifications in addition to any that are naturally present in theActRIIa polypeptides. Such modifications include, but are not limitedto, acetylation, carboxylation, glycosylation, phosphorylation,lipidation, and acylation. As a result, the modified ActRIIapolypeptides may contain non-amino acid elements, such as polyethyleneglycols, lipids, poly- or mono-saccharide, and phosphates. Effects ofsuch non-amino acid elements on the functionality of an ActRIIapolypeptide may be tested as described herein for other ActRIIapolypeptide variants. When an ActRIIa polypeptide is produced in cellsby cleaving a nascent form of the ActRIIa polypeptide,post-translational processing may also be important for correct foldingand/or function of the protein. Different cells (such as CHO, HeLa,MDCK, WI38, NIH-3T3 or HEK293) have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of theActRIIa polypeptides.

In certain aspects, functional variants or modified forms of the ActRIIapolypeptides include fusion proteins having at least a portion of theActRIIa polypeptides and one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (Fc), maltosebinding protein (MBP), or human serum albumin. A fusion domain may beselected so as to confer a desired property. For example, some fusiondomains are particularly useful for isolation of the fusion proteins byaffinity chromatography. For the purpose of affinity purification,relevant matrices for affinity chromatography, such as glutathione-,amylase-, and nickel- or cobalt-conjugated resins are used. Many of suchmatrices are available in “kit” form, such as the Pharmacia GSTpurification system and the QIAexpress™ system (Qiagen) useful with(HIS₆ (SEQ ID NO: 17)) fusion partners. As another example, a fusiondomain may be selected so as to facilitate detection of the ActRIIapolypeptides. Examples of such detection domains include the variousfluorescent proteins (e.g., GFP) as well as “epitope tags,” which areusually short peptide sequences for which a specific antibody isavailable. Well known epitope tags for which specific monoclonalantibodies are readily available include FLAG, influenza virushaemagglutinin (HA), and c-myc tags. In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain preferred embodiments, an ActRIIapolypeptide is fused with a domain that stabilizes the ActRIIapolypeptide in vivo (a “stabilizer” domain). By “stabilizing” is meantanything that increases serum half life, regardless of whether this isbecause of decreased destruction, decreased clearance by the kidney, orother pharmacokinetic effect. Fusions with the Fc portion of animmunoglobulin are known to confer desirable pharmacokinetic propertieson a wide range of proteins. Likewise, fusions to human serum albumincan confer desirable properties. Other types of fusion domains that maybe selected include multimerizing (e.g., dimerizing, tetramerizing)domains and functional domains (that confer an additional biologicalfunction).

As a specific example, the present invention provides methods fortreating or preventing breast cancer using a fusion protein comprising asoluble extracellular domain of ActRIIa fused to an Fc domain (e.g., SEQID NO: 6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

Optionally, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain. It is generally understood that an Fc domain may include smalleror larger portions of the constant region of an immunoglobulin, providedthat the resulting “Fc domain” retains the ability to dimerizecovalently through a disulfide linkage and forms a relative stable,soluble protein.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRIIa polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ActRIIa polypeptide. The ActRIIapolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In certain embodiments, ActRIIa polypeptides useful in accordance withthe methods described herein may contain one or more modifications thatare capable of stabilizing the ActRIIa polypeptides. For example, suchmodifications enhance the in vitro half life of the ActRIIapolypeptides, enhance circulatory half life of the ActRIIa polypeptidesor reducing proteolytic degradation of the ActRIIa polypeptides. Suchstabilizing modifications include, but are not limited to, fusionproteins (including, for example, fusion proteins comprising an ActRIIapolypeptide and a stabilizer domain), modifications of a glycosylationsite (including, for example, addition of a glycosylation site to anActRIIa polypeptide), and modifications of carbohydrate moiety(including, for example, removal of carbohydrate moieties from anActRIIa polypeptide). As used herein, the term “stabilizer domain” notonly refers to a fusion domain (e.g., Fc) as in the case of fusionproteins, but also includes nonproteinaceous modifications such as acarbohydrate moiety, or nonproteinaceous moiety, such as polyethyleneglycol.

In certain embodiments, the methods described herein utilize isolatedand/or purified forms of the ActRIIa polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins. ActRIIapolypeptides will generally be produced by expression from recombinantnucleic acids.

3. Nucleic Acids Encoding ActRIIa Polypeptides

Provided herein are isolated and/or recombinant nucleic acids encodingany of the ActRIIa polypeptides (e.g., soluble ActRIIa polypeptides),including fragments, functional variants and fusion proteins disclosedherein. For example, SEQ ID NO: 4 encodes the naturally occurring humanActRIIa precursor polypeptide, while SEQ ID NO: 5 encodes the processedextracellular domain of ActRIIa. The subject nucleic acids may besingle-stranded or double stranded. Such nucleic acids may be DNA or RNAmolecules. These nucleic acids may be used, for example, in methods formaking ActRIIa polypeptides or as direct therapeutic agents (e.g., in agene therapy approach).

In certain aspects, the subject nucleic acids encoding ActRIIapolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 4 or 5. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants.

In certain embodiments, the invention provides methods for treating orpreventing breast cancer using isolated or recombinant nucleic acidsequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 4 or 5. One of ordinary skill in the art willappreciate that nucleic acid sequences complementary to SEQ ID NO: 4 or5, and variants of SEQ ID NO: 4 or 5 are also within the scope of thisinvention. In further embodiments, the nucleic acid sequences describedherein can be isolated, recombinant, and/or fused with a heterologousnucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids useful in accordance with themethods described herein also include nucleotide sequences thathybridize under highly stringent conditions to the nucleotide sequencedesignated in SEQ ID NO: 4 or 5, complement sequence of SEQ ID NO: 4 or5, or fragments thereof. One of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the methods describedherein utilize nucleic acids which hybridize under low stringencyconditions of 6×SSC at room temperature followed by a wash at 2×SSC atroom temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 4 or 5 due to degeneracy in the genetic code are alsocontemplated for use in accordance with the methods described herein.For example, a number of amino acids are designated by more than onetriplet. Codons that specify the same amino acid, or synonyms (forexample, CAU and CAC are synonyms for histidine) may result in “silent”mutations which do not affect the amino acid sequence of the protein.However, it is expected that DNA sequence polymorphisms that do lead tochanges in the amino acid sequences of the subject proteins will existamong mammalian cells. One skilled in the art will appreciate that thesevariations in one or more nucleotides (up to about 3-5% of thenucleotides) of the nucleic acids encoding a particular protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this invention.

In certain embodiments, the recombinant nucleic acids described hereinmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects, the methods described herein utilize an expressionvector comprising a nucleotide sequence encoding an ActRIIa polypeptidethat is operably linked to at least one regulatory sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe ActRIIa polypeptide. Accordingly, the term regulatory sequenceincludes promoters, enhancers, and other expression control elements.Exemplary regulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology, Academic Press, San Diego, Calif.(1990). For instance, any of a wide variety of expression controlsequences that control the expression of a DNA sequence when operativelylinked to it may be used in these vectors to express DNA sequencesencoding an ActRIIa polypeptide. Such useful expression controlsequences, include, for example, the early and late promoters of SV40,tet promoter, adenovirus or cytomegalovirus immediate early promoter,RSV promoters, the lac system, the trp system, the TAC or TRC system, T7promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage lambda, the control regions forfd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS, thepromoters of the yeast α-mating factors, the polyhedron promoter of thebaculovirus system and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof. It should be understood that the design ofthe expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

A recombinant nucleic acid described herein can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant ActRIIa polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRIIa polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRIIa polypeptides in cells propagated in culture,e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 4 or 5)for one or more of the subject ActRIIa polypeptides. The host cell maybe any prokaryotic or eukaryotic cell. For example, an ActRIIapolypeptide described herein may be expressed in bacterial cells such asE. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art.

Also provided herein are methods of producing the subject ActRIIapolypeptides. For example, a host cell transfected with an expressionvector encoding an ActRIIa polypeptide can be cultured under appropriateconditions to allow expression of the ActRIIa polypeptide to occur. TheActRIIa polypeptide may be secreted and isolated from a mixture of cellsand medium containing the ActRIIa polypeptide. Alternatively, theActRIIa polypeptide may be retained cytoplasmically or in a membranefraction and the cells harvested, lysed and the protein isolated. A cellculture includes host cells, media and other byproducts. Suitable mediafor cell culture are well known in the art. The subject ActRIIapolypeptides can be isolated from cell culture medium, host cells, orboth, using techniques known in the art for purifying proteins,including ion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, immunoaffinity purification withantibodies specific for particular epitopes of the ActRIIa polypeptidesand affinity purification with an agent that binds to a domain fused tothe ActRIIa polypeptide (e.g., a protein A column may be used to purifyan ActRIIa-Fc fusion). In a preferred embodiment, the ActRIIapolypeptide is a fusion protein containing a domain which facilitatesits purification. In a preferred embodiment, purification is achieved bya series of column chromatography steps, including, for example, threeor more of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. Asdemonstrated herein, ActRIIa-hFc protein was purified to a purityof >98% as determined by size exclusion chromatography and >95% asdetermined by SDS PAGE. This level of purity was sufficient to achievedesirable results in mice, rats and non-human primates.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIapolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRIIa polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Alternative Activin and ActRIIa Antagonists

The present disclosure relates to methods for treating or preventingbreast cancer using antagonists of activin-ActRIIa signaling. Althoughsoluble ActRIIa polypeptides, and particularly ActrIIa-Fc, are preferredantagonists, and although such antagonists may affect breast cancer cellgrowth or survival through a mechanism other than activin antagonism(e.g., activin inhibition may be an indicator of the tendency of anagent to inhibit the activities of a spectrum of molecules, including,perhaps, other members of the TGF-beta superfamily, and such collectiveinhibition may lead to the desired effect on breast cancer cell growthor survival), other types of activin-ActRIIa antagonists are expected tobe useful, including anti-activin (e.g., activin β_(A), β_(B), β_(C) andβ_(E)) antibodies, anti-ActRIIa antibodies, antisense, RNAi or ribozymenucleic acids that inhibit the production of ActRIIa and otherinhibitors of activin or ActRIIa, particularly those that disruptactivin-ActRIIa binding. In certain embodiments, antagonists specificfor activin B (e.g., anti-activin B antibodies) are useful in themethods of the present invention.

An antibody that is specifically reactive with an ActRIIa polypeptide(e.g., a soluble ActRIIa polypeptide) and which either bindscompetitively to ligand with the ActRIIa polypeptide or otherwiseinhibits ActRIIa-mediated signaling may be used as an antagonist ofActRIIa polypeptide activities. Likewise, an antibody that isspecifically reactive with an activin β_(A), β_(B), β_(C) or β_(E)polypeptide, or any heterodimer thereof, and which disrupts ActRIIabinding may be used as an antagonist.

By using immunogens derived from an ActRIIa polypeptide or an activinpolypeptide, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (see, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the ActRIIa polypeptide, an antigenicfragment which is capable of eliciting an antibody response, or a fusionprotein. Techniques for conferring immunogenicity on a protein orpeptide include conjugation to carriers or other techniques well knownin the art. An immunogenic portion of an ActRIIa or activin polypeptidecan be administered in the presence of adjuvant. The progress ofimmunization can be monitored by detection of antibody titers in plasmaor serum. Standard ELISA or other immunoassays can be used with theimmunogen as antigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of anActRIIa polypeptide, antisera can be obtained and, if desired,polyclonal antibodies can be isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an ActRIIapolypeptide and monoclonal antibodies isolated from a culture comprisingsuch hybridoma cells. Antibodies may also be generated by screeninglibraries (e.g., phage display libraries) of antibody variable domainsor Fab fragments to identify binders that bind to the selected antigen(e.g., activins or ActRIIa). This in vitro approach is often useful withproteins that are highly conserved between mammals, particularly miceand humans.

The term “antibody” as used herein is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includesfragments or domains of immunoglobulins which are reactive with aselected antigen. Antibodies can be fragmented using conventionaltechniques and the fragments screened for utility and/or interactionwith a specific epitope of interest. Thus, the term includes segments ofproteolytically-cleaved or recombinanrtly-prepared portions of anantibody molecule that are capable of selectively reacting with acertain protein. Non-limiting examples of such proteolytic and/orrecombinant fragments include Fab, F(ab′)₂, Fab′, Fv, and single chainantibodies (scFv) containing a V[L] and/or V[H] domain joined by apeptide linker. The scFv's may be covalently or non-covalently linked toform antibodies having two or more binding sites. The term antibody alsoincludes polyclonal, monoclonal, or other purified preparations ofantibodies and recombinant antibodies. The term “recombinant antibody”,means an antibody, or antigen binding domain of an immunoglobulin,expressed from a nucleic acid that has been constructed using thetechniques of molecular biology, such as a humanized antibody or a fullyhuman antibody developed from a single chain antibody. Single domain andsingle chain antibodies are also included within the term “recombinantantibody”.

In certain embodiments, the methods described herein may utilize anantibody, such as, for example, a monoclonal antibody. Also provided aremethods for generating novel antibodies. For example, a method forgenerating a monoclonal antibody that binds specifically to an ActRIIapolypeptide or activin polypeptide may comprise administering to a mousean amount of an immunogenic composition comprising the antigenpolypeptide effective to stimulate a detectable immune response,obtaining antibody-producing cells (e.g., cells from the spleen) fromthe mouse and fusing the antibody-producing cells with myeloma cells toobtain antibody-producing hybridomas, and testing the antibody-producinghybridomas to identify a hybridoma that produces a monocolonal antibodythat binds specifically to the antigen. Once obtained, a hybridoma canbe propagated in a cell culture, optionally in culture conditions wherethe hybridoma-derived cells produce the monoclonal antibody that bindsspecifically to the antigen. The monoclonal antibody may be purifiedfrom the cell culture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., an ActRIIa polypeptide) and other antigens that are notof interest that the antibody is useful for, at minimum, detecting thepresence of the antigen of interest in a particular type of biologicalsample. In certain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody: antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ M or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore™ binding assay, Biacore A B, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

Examples of categories of nucleic acid compounds that are activin orActRIIa antagonists include antisense nucleic acids, RNAi constructs andcatalytic nucleic acid constructs. A nucleic acid compound may be singleor double stranded. A double stranded compound may also include regionsof overhang or non-complementarity, where one or the other of thestrands is single stranded. A single stranded compound may includeregions of self-complementarity, meaning that the compound forms aso-called “hairpin” or “stem-loop” structure, with a region of doublehelical structure. A nucleic acid compound may comprise a nucleotidesequence that is complementary to a region consisting of no more than1000, no more than 500, no more than 250, no more than 100, or no morethan 50, 35, 25, 22, 20, 18 or 15 nucleotides of the full-length ActRIIanucleic acid sequence or activin β_(A), β_(B), β_(C), or β_(E) nucleicacid sequence. The region of complementarity will preferably be at least8 nucleotides, and optionally about 18 to 35 nucleotides. A region ofcomplementarity may fall within an intron, a coding sequence or anoncoding sequence of the target transcript, such as the coding sequenceportion. Generally, a nucleic acid compound will have a length of about8 to about 500 nucleotides or base pairs in length, and optionally thelength will be about 14 to about 50 nucleotides. A nucleic acid may be aDNA (particularly for use as an antisense), RNA or RNA:DNA hybrid. Anyone strand may include a mixture of DNA and RNA, as well as modifiedforms that cannot readily be classified as either DNA or RNA. Likewise,a double stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and anyone strand may also include a mixture of DNA and RNA, as well asmodified forms that cannot readily be classified as either DNA or RNA. Anucleic acid compound may include any of a variety of modifications,including one or modifications to the backbone (the sugar-phosphateportion in a natural nucleic acid, including internucleotide linkages)or the base portion (the purine or pyrimidine portion of a naturalnucleic acid). An antisense nucleic acid compound will preferably have alength of about 15 to about 30 nucleotides and will often contain one ormore modifications to improve characteristics such as stability in theserum, in a cell or in a place where the compound is likely to bedelivered, such as the stomach in the case of orally delivered compoundsand the lung for inhaled compounds. In the case of an RNAi construct,the strand complementary to the target transcript will generally be RNAor modifications thereof. The other strand may be RNA, DNA or any othervariation. The duplex portion of double stranded or single stranded“hairpin” RNAi construct will generally have a length of 18 to 40nucleotides in length and optionally about 21 to 23 nucleotides inlength, so long as it serves as a Dicer substrate. Catalytic orenzymatic nucleic acids may be ribozymes or DNA enzymes and may alsocontain modified forms. Nucleic acid compounds may inhibit expression ofthe target by about 50%, 75%, 90% or more when contacted with cellsunder physiological conditions and at a concentration where a nonsenseor sense control has little or no effect. Preferred concentrations fortesting the effect of nucleic acid compounds are 1, 5 and 10 micromolar.Nucleic acid compounds may also be tested for effects on, for example,the proliferation or survival of breast cancer cells or breast tumors.

5. Screening Assays

In certain aspects, the present invention relates to the use of ActRIIapolypeptides (e.g., soluble ActRIIa polypeptides) and activinpolypeptides to identify compounds (agents) which are agonist orantagonists of the activin-ActRIIa signaling pathway. Compoundsidentified through this screening can be tested to assess their abilityto modulate the growth or survival of cancer cells, particularly breastcancer cells, in vivo or in vitro. These compounds can be tested, forexample, in animal models such as mouse xenograft models. One usefulanimal model is the murine MDA-MB231 breast cancer model; MDA-MB231cells are hormone-independent and are prone to metastasize to the bone.Other animal models of breast cancer can be generated, for example, byimplanting rat neuroblastoma cells (from which the neu oncogene wasinitially isolated), or neu-transformed NIH-3T3 cells into nude mice,essentially as described by Drebin et al. Proc. Nat. Acad. Sci. USA, 83:9129-9133 (1986).

There are numerous approaches to screening for therapeutic agents fortreating or preventing breast cancer by targeting activin and ActRIIasignaling. In certain embodiments, high-throughput screening ofcompounds can be carried out to identify agents that perturb activin orActRIIa-mediated effects on a selected cell line. In certainembodiments, the assay is carried out to screen and identify compoundsthat specifically inhibit or reduce binding of an ActRIIa polypeptide toactivin. Alternatively, the assay can be used to identify compounds thatenhance binding of an ActRIIa polypeptide to activin. In a furtherembodiment, the compounds can be identified by their ability to interactwith an activin or ActRIIa polypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,test compounds (agents) may be created by any combinatorial chemicalmethod. Alternatively, the subject compounds may be naturally occurringbiomolecules synthesized in vivo or in vitro. Compounds (agents) to betested for their ability to act as modulators of tissue growth can beproduced, for example, by bacteria, yeast, plants or other organisms(e.g., natural products), produced chemically (e.g., small molecules,including peptidomimetics), or produced recombinantly. Test compoundscontemplated herein include non-peptidyl organic molecules, peptides,polypeptides, peptidomimetics, sugars, hormones, and nucleic acidmolecules. In a specific embodiment, the test agent is a small organicmolecule having a molecular weight of less than about 2,000 Daltons.

Test compounds can be provided as single, discrete entities, or providedin libraries of greater complexity, such as made by combinatorialchemistry. These libraries can comprise, for example, alcohols, alkylhalides, amines, amides, esters, aldehydes, ethers and other classes oforganic compounds. Presentation of test compounds to the test system canbe in either an isolated form or as mixtures of compounds, especially ininitial screening steps. Optionally, the compounds may be optionallyderivatized with other compounds and have derivatizing groups thatfacilitate isolation of the compounds. Non-limiting examples ofderivatizing groups include biotin, fluorescein, digoxygenin, greenfluorescent protein, isotopes, polyhistidine, magnetic beads,glutathione S transferase (GST), photoactivatible crosslinkers or anycombinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIapolypeptide and activin.

Merely to illustrate, in an exemplary screening assay, the compound ofinterest is contacted with an isolated and purified ActRIIa polypeptidewhich is ordinarily capable of binding to activin. To the mixture of thecompound and ActRIIa polypeptide is then added a composition containingan ActRIIa ligand. Detection and quantification of ActRIIa/activincomplexes provides a means for determining the compound's efficacy atinhibiting (or potentiating) complex formation between the ActRIIapolypeptide and activin. The efficacy of the compound can be assessed bygenerating dose response curves from data obtained using variousconcentrations of the test compound. Moreover, a control assay can alsobe performed to provide a baseline for comparison. For example, in acontrol assay, isolated and purified activin is added to a compositioncontaining the ActRIIa polypeptide, and the formation of ActRIIa/activincomplex is quantitated in the absence of the test compound. It will beunderstood that, in general, the order in which the reactants may beadmixed can be varied, and can be admixed simultaneously. Moreover, inplace of purified proteins, cellular extracts and lysates may be used torender a suitable cell-free assay system.

Complex formation between the ActRIIa polypeptide and activin may bedetected by a variety of techniques. For instance, modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or ³H),fluorescently labeled (e.g., FITC), or enzymatically labeled ActRIIapolypeptide or activin, by immunoassay, or by chromatographic detection.

In certain embodiments, fluorescence polarization assays andfluorescence resonance energy transfer (FRET) assays may be used formeasuring, either directly or indirectly, the degree of interactionbetween an ActRIIa polypeptide and its binding protein. Other suitablemodes of detection include, for example, those based on opticalwaveguides (PCT Publication WO 96/26432 and U.S. Pat. No. 5,677,196),surface plasmon resonance (SPR), surface charge sensors, and surfaceforce sensors.

An interaction trap assay, also known as the “two hybrid assay,” mayalso be used for identifying agents that disrupt or potentiateinteraction between an ActRIIa polypeptide and its binding protein. Seefor example, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel etal. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene8:1693-1696). In a specific embodiment, a reverse two hybrid system maybe used to identify compounds (e.g., small molecules or peptides) thatdissociate interactions between an ActRIIa polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, compounds are identified by their ability tointeract with an ActRIIa or activin polypeptide described herein. Theinteraction between the compound and the ActRIIa or activin polypeptidemay be covalent or non-covalent. For example, such interaction can beidentified at the protein level using in vitro biochemical methods,including photo-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, the compounds may be screened in a mechanism basedassay, such as an assay to detect compounds which bind to an activin orActRIIa polypeptide. This may include a solid phase or fluid phasebinding event. Alternatively, the gene encoding an activin or ActRIIapolypeptide can be transfected with a reporter system (e.g.,β-galactosidase, luciferase, or green fluorescent protein) into a celland screened against the library optionally by a high throughputscreening or with individual members of the library. Other mechanismbased binding assays may be used, for example, binding assays whichdetect changes in free energy. Binding assays can be performed with thetarget fixed to a well, bead or chip or captured by an immobilizedantibody or resolved by capillary electrophoresis. The bound compoundsmay be detected usually using calorimetric or fluorescence or surfaceplasmon resonance.

6. Exemplary Therapeutic Uses

In certain embodiments, the present invention provides methods oftreating or preventing breast cancer in an individual in need thereof byadministering to the individual a therapeutically effective amount of anactivin-ActRIIa antagonist, such as, for example, an ActRIIapolypeptide. These methods may be used for therapeutic as well asprophylactic treatment of humans, particularly females, who have a highrisk for developing breast cancer. As every woman is at risk fordeveloping breast cancer, a woman with a high risk for developing breastcancer is a woman whose risk factors confer a greater probability ofdeveloping the disease compared to the general population or thepopulation of women within a certain age group. Exemplary risk factorsinclude age, family history or genetic makeup, lifestyle habits such asexercise and diet, exposure to radiation or other cancer-causing agents,age at the time the first child was born, genetic changes, and weightgain after menopause.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset of one or moresymptoms or characteristics of the disorder or condition relative to theuntreated control sample. For example, preventing breast cancer mayrefer to the absence of new lesions following treatment, or the absenceor delay of metastatic disease.

The term “treating breast cancer” refers to an improvement of one ormore symptoms or characteristics of the disease relative to an untreatedcontrol or relative to the severity of disease prior to treatment. Theterm does not necessarily require that the patient receiving thetreatment be cured or that the disease be completely eradicated from thepatient. An agent that treats breast cancer may be an agent that reducesthe severity of one or more symptoms or characteristics of the disease.It should be noted that tumor growth and progression is influenced by avariety of factors, including mediators of cell cycle progression andcell division and regulators of cell death, or apoptosis. Accordingly,treating breast cancer may involve a decrease in cancer cellproliferation or a decrease in the rate of cell division. Alternativelyor additionally, treating breast cancer may involve a decrease in cancercell survival, an increase in apoptosis or a diminished occurrence orseverity of metastatic breast cancer, particularly metastatic breastcancer of the bone. Accordingly, in certain embodiments, treating breastcancer may involve both a decrease in cell division and an increase incell death. Regardless of mechanism, the effectiveness of an agent intreating breast cancer may be determined by observable metrics, such asa lower number of cancer cells compared to a control (either due todecreased proliferation, increased apoptosis, or both), or a decrease intumor size compared to a control. Therefore treating breast cancer orinhibiting tumor or cancer cell growth is intended to be neutral as tothe mechanism by which such a change occurs. Both prevention andtreatment may be discerned in the diagnosis provided by a physician orother health care provider and the analysis of the intended result ofadministration of the therapeutic agent.

When observing the effects of the subject antagonists on breast cancerprogression in humans, an effect may be evaluated by a decrease ordisappearance of measurable disease, and/or the absence of new lesionsor the prevention of metastases. For example, activin-ActRIIaantagonists may significantly reduce or delay breast cancer progressionin patients with both noninvasive and invasive breast cancer. Inaddition, the antagonists may prevent or reduce the risk of developingbreast cancer in healthy women with risk factors for the disease. Theantagonists may also reduce the risk of breast cancer recurrence inpatients with a history of the disease.

Accordingly, activin-ActRIIa antagonists may be used to prevent or delaythe onset of breast cancer in individuals considered to be at risk fordeveloping the disease, and such antagonists may be used in selectedpatient populations. Examples of appropriate patient populations includepatients with a family history of breast and ovarian cancer, such asfemale patients where a mother or sister has been diagnosed with thedisease. Patients that have mutations in the BRCA1/2 genes or othergenes shown to predispose women to breast and ovarian cancer are alsoincluded. In one embodiment, a patient considered to be at high risk fordeveloping breast cancer but who has not been diagnosed with the diseaseis treated with an activin-ActRIIa antagonist. Such treatment may beginwhen the patient reaches the age of 30, 35, or 40, or when a femalepatient is not trying to conceive (i.e., the patient does not plan tonurse an infant) or has reached menopause. In particular, data presentedherein demonstrates that activin-ActRIIa antagonists inhibit themetastatic spread of a breast cancer cell line introduced into thegeneral circulation, demonstrating that such antagonists may be usefulin preventing the metastases of breast tumors. Such compounds would beuseful in treating any patient that has been diagnosed with breastcancer or suspected of having breast cancer. Additionally, patients thatare considering having a preventative, or elective, mastectomy due to anelevated risk of developing a breast tumor may elect instead or inaddition to take an activin-ActRIIa antagonist to diminish the risk ofmetastatic spread of undetected tumors.

Activin-ActRIIa antagonists disclosed herein, and particularlyActRIIa-Fc proteins, may be used to treat or prevent breast cancer in apatient, including patients with solid tumors as well as patients withmetastatic cancer. Activin-ActRIIa antagonists may also be administeredto human subjects with precancerous or benign lesions of the breast orwith any abnormal proliferative lesions including typical hyperplasia,atypical hyperplasia, and noninvasive or in situ carcinoma. Theantagonists of the present disclosure are also useful in the treatmentor prevention of both hormone-dependent or hormone-responsive cancers(e.g., estrogen receptor positive cancers) and hormone-independentcancers (e.g., estrogen receptor negative or estrogen receptor mutantcancers). Activin-ActRIIa antagonists are also useful as therapeuticagents for cancers in which growth factors or oncogenes are activated(e.g., breast cancers in which c-erbB-2 (also known as HER-2/Neu)tyrosine kinase is expressed). Activin-ActRIIa antagonists may prove tobe particularly useful in tumors that express elevated (relative tonormal breast tissue-derived cells) levels of activin (e.g., A, AB or B)or elevated levels of ActRIIa or ActRIIb.

The present invention recognizes that the effectiveness of conventionalcancer therapies (e.g., chemotherapy, radiation therapy, phototherapy,immunotherapy, and surgery) can be enhanced through the use of thesubject antagonists. Accordingly, activin-ActRIIa antagonists may beused in combination therapies for the treatment, prevention, ormanagement of breast cancer. The antagonists may be administered topatients in combination with radiation and/or surgical treatment as wellas with cytotoxic chemotherapy and/or endocrine therapies. Suchcombination treatments may work synergistically and allow reduction ofdosage of each of the individual treatments, thereby reducing thedetrimental side effects exerted by each treatment at higher dosages. Inother instances, malignancies that are refractory to a treatment mayrespond to a combination therapy of two or more different treatments.Accordingly, the disclosure relates to the administration of anactivin-ActRIIa antagonist in combination with another conventionalanti-neoplastic agent, either concomitantly or sequentially, in order toenhance the therapeutic effect of the anti-neoplastic agent or overcomecellular resistance to such anti-neoplastic agent.

Pharmaceutical compounds that may be used for combinatory anti-tumortherapy include, merely to illustrate: aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

These chemotherapeutic anti-tumor compounds may be categorized by theirmechanism of action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (vascular endothelial growth factor (VEGF) inhibitors,fibroblast growth factor (FGF) inhibitors); angiotensin receptorblocker; nitric oxide donors; anti-sense oligonucleotides; antibodies(trastuzumab); cell cycle inhibitors and differentiation inducers(tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin(adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin,eniposide, epirubicin, etoposide, idarubicin and mitoxantrone,topotecan, irinotecan), corticosteroids (cortisone, dexamethasone,hydrocortisone, methylpednisolone, prednisone, and prenisolone); growthfactor signal transduction kinase inhibitors; mitochondrial dysfunctioninducers and caspase activators; and chromatin disruptors.

In certain embodiments, pharmaceutical compounds that may be used forcombinatory therapy include anti-angiogenesis agents such as (1)inhibitors of release of “angiogenic molecules,” such as bFGF (basicfibroblast growth factor); (2) neutralizers of angiogenic molecules,such as an anti-βbFGF antibodies; and (3) inhibitors of endothelial cellresponse to angiogenic stimuli, including collagenase inhibitor,basement membrane turnover inhibitors, angiostatic steroids,fungal-derived angiogenesis inhibitors, platelet factor 4,thrombospondin, arthritis drugs such as D-penicillamine and goldthiomalate, vitamin D3 analogs, alpha-interferon, and the like. Foradditional proposed inhibitors of angiogenesis, see Blood et al., Bioch.Biophys. Acta., 1032:89-118 (1990), Moses et al., Science, 248:1408-1410(1990), Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S. Pat. Nos.5,092,885, 5,112,946, 5,192,744, 5,202,352, and 6573256. In addition,there are a wide variety of compounds that can be used to inhibitangiogenesis, for example, peptides or agents that block theVEGF-mediated angiogenesis pathway, endostatin protein or derivatives,lysine binding fragments of angiostatin, melanin or melanin-promotingcompounds, plasminogen fragments (e.g., Kringles 1-3 of plasminogen),tropoin subunits, antagonists of vitronectin αvβ3, peptides derived fromSaposin B, antibiotics or analogs (e.g., tetracycline, or neomycin),dienogest-containing compositions, compounds comprising a MetAP-2inhibitory core coupled to a peptide, the compound EM-138, chalcone andits analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos.6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810,6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, 6,538,103,6,544,758, 6,544,947, 6,548,477, 6,559,126, and 6,569,845.

Depending on the nature of the combinatory therapy, administration ofthe therapeutic antagonists of the invention may be continued while theother therapy is being administered and/or thereafter. Administration ofthe antagonists described herein may be made in a single dose, or inmultiple doses. In some instances, administration of the antagonists iscommenced at least several days prior to the conventional therapy, whilein other instances, administration is begun either immediately before orat the time of the administration of the conventional therapy.

7. Pharmaceutical Compositions

In certain embodiments, activin-ActRIIa antagonists described herein areformulated with a pharmaceutically acceptable carrier. For example, anActRIIa polypeptide can be administered alone or as a component of apharmaceutical formulation (therapeutic composition). The subjectantagonists may be formulated for administration in any convenient wayfor use in human or veterinary medicine.

In certain embodiments, the methods for treating or preventing or breastcancer as described herein include administering the compositionsystemically, or locally as an implant or device. When administered, thetherapeutic composition for use in this invention is, of course, in apyrogen-free, physiologically acceptable form. Therapeutically usefulagents other than the activin-ActRIIa antagonists which may alsooptionally be included in the composition as described above, may beadministered simultaneously or sequentially with the subject antagonistsin the methods of the invention.

Typically, activin-ActRIIa antagonists will be administered parentally.Pharmaceutical compositions suitable for parenteral administration maycomprise one or more ActRIIa polypeptides in combination with one ormore pharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site (e.g., mammary epithelium). In certainembodiments, compositions described herein may include a matrix capableof delivering one or more therapeutic compounds (e.g., ActRIIapolypeptides) to a target tissue site (e.g., mammary epithelium),providing a structure for the developing tissue and optimally capable ofbeing resorbed into the body. For example, the matrix may provide slowrelease of the ActRIIa polypeptides. Such matrices may be formed ofmaterials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, antagonists described herein can be administeredorally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An antagonist may also be administered asa bolus, electuary or paste.

In solid dosage forms for oral administration. (capsules, tablets,pills, dragees, powders, granules, and the like), one or moretherapeutic antagonists may be mixed with one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate,and/or any of the following: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as, for example, carboxymethylcellulose, alginates, gelatin,polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such asglycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Compositions useful in accordance with the methods described herein mayalso contain adjuvants, such as preservatives, wetting agents,emulsifying agents and dispersing agents. Prevention of the action ofmicroorganisms may be ensured by the inclusion of various antibacterialand antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid, and the like. It may also be desirable to include isotonicagents, such as sugars, sodium chloride, and the like into thecompositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption, such as aluminum monostearate and gelatin.

It is understood that the dosage regimen suitable for treating orpreventing breast cancer will be determined by the attending physicianconsidering various factors which modify the action of the subjectcompounds of the invention (e.g., ActRIIa polypeptides). The variousfactors include, but are not limited to, the patient's age, sex, anddiet, the severity of the disease, time of administration, and otherclinical factors. The addition of other known growth factors to thefinal composition may also affect the dosage. Progress can be monitoredby periodic assessment of various factors including but not limited totumor size, stage, or histological grade, estrogen or progesteronereceptor status, angioinvasion, and regional lymph node metastasis. Theclinician may also monitor markers such as levels of the proteinuPA/PAI1-high levels of uPA and PAI1 are associated with a high risk ofmetastasis—and Her-2 gene amplification and/or protein expression, whichis also associated with metastasis (Weigelt et al. 2005 Nat. Rev. Cancer5: 591-602). Gene expression profiling may also prove to be helpful inmonitoring disease progression (van 't Veer et al. 2002 Nature 415:530-536 and van de Vijver et al. 2002 N. Engl. J. Med. 347: 1999-2009).

In certain embodiments, the present invention also provides methods fortreating or preventing breast cancer that involve gene therapy for thein vivo production of ActRIIa polypeptides. Such therapy would achieveits therapeutic effect by introduction of the ActRIIa polynucleotidesequences into cells or tissues involved in breast cancer, such as, forexample, mammary epithelial cells. Delivery of ActRIIa polynucleotidesequences can be achieved using a recombinant expression vector such asa chimeric virus or a colloidal dispersion system. Preferred fortherapeutic delivery of ActRIIa polynucleotide sequences is the use oftargeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus suchas a retrovirus. The retroviral vector may be a derivative of a murineor avian retrovirus. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Anumber of additional retroviral vectors can incorporate multiple genes.All of these vectors can transfer or incorporate a gene for a selectablemarker so that transduced cells can be identified and generated.Retroviral vectors can be made target-specific by attaching, forexample, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing the ActRIIapolynucleotide.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRIIa polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 ActRIIa-Fc Fusion Proteins

Applicants constructed a soluble ActRIIa fusion protein that has theextracellular domain of human ActRIIa fused to a human or mouse Fcdomain with a minimal linker in between. The constructs are referred toas ActRIIa-hFc and ActRIIa-mFc, respectively.

ActRIIa-hFc is shown below as purified from CHO cell lines (SEQ ID NO:7):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO celllines. Three different leader sequences were considered:

(i) Honey bee melitin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 8),

(ii) Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ IDNO: 9), and

(iii) Native: MGAAAKLAFAVFLISCSSGA (SEQ ID NO: 10).

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

(SEQ ID NO: 13) MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence:

(SEQ ID NO: 14) ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGAGAATTC

Both ActRIIa-hFc and ActRIIa-mFc were remarkably amenable to recombinantexpression. As shown in FIG. 1, the protein was purified as a single,well-defined peak of protein. N-terminal sequencing revealed a singlesequence of—ILGRSETQE (SEQ ID NO: 11). Purification could be achieved bya series of column chromatography steps, including, for example, threeor more of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. TheActRIIa-hFc protein was purified to a purity of >98% as determined bysize exclusion chromatography and >95% as determined by SDS PAGE.

ActRIIa-hFc and ActRIIa-mFc showed a high affinity for ligands,particularly activin A. GDF-11 or Activin A (“ActA”) were immobilized ona Biacore CM5 chip using standard amine coupling procedure. ActRIIa-hFcand ActRIIa-mFc proteins were loaded onto the system, and binding wasmeasured. ActRIIa-hFc bound to activin with a dissociation constant(K_(D)) of 5×10⁻¹², and the protein bound to GDF11 with a K_(D) of9.96×10⁻⁹. See FIG. 2. ActRIIa-mFc behaved similarly.

The ActRIIa-hFc was very stable in pharmacokinetic studies. Rats weredosed with 1 mg/kg, 3 mg/kg or 10 mg/kg of ActRIIa-hFc protein andplasma levels of the protein were measured at 24, 48, 72, 144 and 168hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg or 30mg/kg. In rats, ActRIIa-hFc had an 11-14 day serum half life andcirculating levels of the drug were quite high after two weeks (11μg/ml, 110 μg/ml or 304 μg/ml for initial administrations of 1 mg/kg, 10mg/kg or 30 mg/kg, respectively.) In cynomolgus monkeys, the plasma halflife was substantially greater than 14 days and circulating levels ofthe drug were 25 μg/ml, 304 μg/ml or 1440 μg/ml for initialadministrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.

Example 2 Characterization of an ActRIIa-hFc Protein

ActRIIa-hFc fusion protein was expressed in stably transfected CHO-DUKXB11 cells from a pAID4 vector (SV40 ori/enhancer, CMV promoter), using atissue plasminogen leader sequence of SEQ ID NO:9. The protein, purifiedas described above in Example 1, had a sequence of SEQ ID NO:7. The Fcportion is a human IgG1 Fc sequence, as shown in SEQ ID NO:7. Sialicacid analysis showed that the protein contained, on average, betweenabout 1.5 and 2.5 moles of sialic acid per molecule of ActRIIa-hFcfusion protein.

This purified protein showed a remarkably long serum half-life in allanimals tested, including a half-life of 25-32 days in human patients(see Example 3, below). The CHO cell expressed material has a higheraffinity for activin B ligand than that reported for an ActRIIa-hFcfusion protein expressed in human 293 cells (del Re et al., J Biol.Chem. 2004 Dec. 17; 279(51):53126-35.). Additionally, the use of the tPaleader sequence provided greater production than other leader sequencesand, unlike ActRIIa-Fc expressed with a native leader, provided a highlypure N-terminal sequence. Use of the native leader sequence resulted intwo major species of ActRIIa-Fc, each having a different N-terminalsequence.

Example 3 Human Clinical Trial

The protein described in Example 2 was administered to human patients ina randomized, double-blind, placebo-controlled study that was conductedto evaluate, primarily, the safety of the protein in healthy,postmenopausal women. Forty-eight subjects were randomized in cohorts of6 to receive either a single dose of ActRIIa-hFc or placebo (5 active:1placebo). Dose levels ranged from 0.01 to 3.0 mg/kg intravenously (IV)and 0.03 to 0.1 mg/kg subcutaneously (SC). All subjects were followedfor 120 days. Subjects were excluded from study participation if theytook medications affecting bone metabolism within 6 months of studyentry. Safety evaluations were conducted following each cohort todetermine dose escalation. In addition to pharmacokinetic (PK) analyses,the biologic activity of ActRIIa-hFc was also assessed by measurement ofbiochemical markers of bone formation and resorption, and FSH levels. Noserious adverse events were reported in this study. Adverse events (AEs)were generally mild and transient. Preliminary analysis of AEs includedheadache, elevated laboratory values, cold symptoms, emesis or vomiting,intravenous infiltration, and hematoma at injection site.

PK analysis of ActRIIa-hFc displayed a linear profile with dose, and amean half-life of approximately 25-32 days. The area-under-curve (AUC)for ActRIIa-hFc was linearly related to dose, and the absorption afterSC dosing was essentially complete. These data indicate that SC is adesirable approach to dosing because it provides equivalentbioavailability and serum-half life for the drug while avoiding thespike in serum concentrations of drug associated with the first few daysof IV dosing. ActRIIa-hFc caused a rapid, sustained dose-dependentincrease in serum levels of bone-specific alkaline phosphatase (BAP),which is a marker for anabolic bone growth, and a dose-dependentdecrease in C-terminal type 1 collagen telopeptide andtartrate-resistant acid phosphatase Sb levels, which are markers forbone resorption. Other markers, such as P1NP showed inconclusiveresults. BAP levels showed near saturating effects at the highest dosageof drug, indicating that half-maximal effects on this anabolic bonebiomarker could be achieved at a dosage of 0.3 mg/kg, with increasesranging up to 3 mg/kg. Calculated as a relationship of pharmacodynamiceffect to AUC for drug, the EC50 is 51,465 (day*ng/ml). These bonebiomarker changes were sustained for approximately 120 days at thehighest dose levels tested. There was also a dose-dependent decrease inserum FSH levels consistent with inhibition of activin.

A single dose of ActRIIa-hFc given to healthy postmenopausal women wassafe and well-tolerated for the range of dose levels tested. Theprolonged PK and pharmacodynamic effects suggest that intermittentdosing would be appropriate for future studies. For example, dosing onthe basis of serum half-life could be performed on a monthly basis, oron the order of once every two, three, four, five or six weeks.Additionally, because the pharmacodynamic effect extends far beyond theserum residence of the drug, dosing could be performed on the basis ofthe pharmacodynamic effect, meaning that dosing every three months orevery two, three, four, five, six or even twelve months may be effectiveto produce the desired effect in patients. This clinical trialdemonstrates that, in humans, ActRIIa-hFc is an osteoanabolic agent withbiological evidence of both an increase in bone formation and a decreasein bone resorption.

Example 4 ActRIIa-Fc Ameliorates or Prevents Bone Loss Caused by BreastCancer Metastases

It is estimated that 65 to 75 percent of breast cancers metastasize tothe bone, causing substantial damage to the bone structure, increasingfracture risk and causing pain and other side effects. We tested theeffects of ActRIIa-Fc in a mouse model of breast cancer that hasmetastasized to the bone.

A subline of the human breast cancer cell line MDA-MB-231 (clone 2287,Kang et al. Cancer Cell 2003, vol 3:537-549) was cultured in vitro andcells harvested at a density of 5×10⁶ cells/ml. MDA-MB-231 is a cellline that is highly competent for seeding into bone and causing bonedamage similar to that caused by bone metastases. 10 μl of cells wereinjected into the tibia of 6 week old female athymic nude mice on studyday 0. On study day 10 mice received ActRIIa-mFc (10 mg/kg/twiceweekly/subcutaneous) (n=8) or PBS vehicle (n=7). Disease progression wasassessed by changes in bone mineral density using dual energy x-rayabsorptiometry (PIXIMus) at weekly intervals. Mice were treated withActRIIa-mFc for 4 weeks and then sacrificed and tibae (both tumorinjected and untumored) were collected from each animal. Tibiae werethen processed and prepared for microcomputed tomography (microCT) andhistololgical analysis.

Intratibial injection of MDA-MB-231 cells into athymic nude micepromoted the development of osteolytic bone lesions in the injectedtibia compared to the contralateral leg. MicroCT analysis of theproximal tibia, demonstrated a 62%, reduction in cancellous bone volumein the MDA-MB-231 bearing tibiae compared to the untumored tibia in PBSvehicle treated mice. ActRIIa-mFc treatment led to an increase of 70% or147% in the naïve or tumor bearing tibia respectively compared tovehicle (P<0.01 for both). The tumor bearing tibiae of ActRIIa-mFctreated mice had a similar cancellous bone density as the naïve tibiaeof the VEH treated mice (p=0.39).

Thus, ActRIIa-mFc is able to eliminate the bone damage associated withthe presence of breast tumor cells in the bone.

Example 5 ActRIIa-Fc Reduces Breast Cancer Metastases and PromotesSurvival

As a model of metastatic disease, MDA-MB-231 cells can be introducedinto mice by intracardiac injection. Cells injected into the leftventricle will migrate through the bloodstream and form metastaticlesions at distal sites. A derivative cell line MDA-MB-231-luc-D3H2LN(Caliper Life Sciences) is a luciferase expressing cell line that allowsfor non-invasive monitoring of metastatic tumor formation usingbiophotonic imaging technology (Caliper Life Sciences). This model wasused to evaluate the potential of ActRIIa-mFc to diminish the formationof metastatic breast cancer lesions.

MDA-MB-231-luc-D3H2LN cells were introduced by intracardiac injectioninto twenty-six athymic nude mice. Fourteen of the mice were treatedwith vehicle (Phosphate buffered saline-PBS) and twelve were treatedwith ActRIIa-mFc (10 mg/kg, twice weekly, subcutaneous injection)starting two weeks prior to tumor administration and continuing throughthe course of the study. An additional nine mice were mock injected withcells and treated with ActRIIa-mFc. Mice were periodically anaesthetizedand visualized for bioluminescent emission to detect the formation ofmetastatic progression.

The ActRIIa-mFc treatment group showed substantially reduced developmentof metastatic lesions. By week five, twelve of fourteen vehicle treatedmice showed multiple, strong fluorescent signals indicative ofmetastatic spread, while only four of twelve ActRIIa-mFc treated miceshowed similar lesions (FIG. 3). A quantitation of fluorescenceintensity showed a roughly ten-fold decrease in fluorescence signal inthe treated mice.

Moreover, the ActRIIa-mFc treatment markedly increased survival of themice. By study day forty, all (14/14) of the vehicle treated mice haddied or had been euthanized (according to standard procedures for humanetreatment of study animals), while only two (2/12) of the ActRIIa-mFctreated mice had died or had been euthanized. By day forty-five, 3/12ActRIIa-mFc treated mice had died or had been euthanized, and none ofthe mock injected mice had died.

Therefore, ActRIIa-mFc treatment causes a substantial decrease in theformation of metastatic lesions, and promotes survival, in this model ofmetastatic breast cancer. These data indicate that ActRIIa-Fc may beused to treat breast cancer in human patients, particularly inconjunction with therapies, such as surgery, hormone therapy ortraditional chemotherapy which target the primary tumor.

Example 6 Alternative ActRIIa-Fc Proteins

A variety of ActRIIa variants that may be used according to the methodsdescribed herein are described in the International Patent Applicationpublished as WO2006/012627 (see e.g., pp. 55-60), incorporated herein byreference in its entirety. An alternative construct may have a deletionof the C-terminal tail (the final 15 amino acids of the extracellulardomain of ActRIIa. The sequence for such a construct is presented below(Fc portion underlined) (SEQ ID NO: 12):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

We claim:
 1. A method for treating or preventing estrogenreceptor-negative breast cancer in a human patient, the methodcomprising administering to a patient in need thereof an effectiveamount of an ActRIIa-Fc fusion protein, wherein the ActRIIa-Fc fusionprotein comprises a polypeptide comprising an amino acid sequence thatis at least 95% identical to SEQ ID NO:2, and wherein the ActRIIa-Fcfusion protein binds to activin A.
 2. The method of claim 1, wherein theActRIIa-Fc fusion protein has one or more of the followingcharacteristics: a. binds to activin A with a K_(D) of at least 10⁻⁷ M;and b. inhibits ActRIIa signaling in a cell.
 3. The method of claim 2,wherein said ActRIIa-Fc fusion protein comprises one or more modifiedamino acid residues selected from: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, and an amino acid conjugated to a lipidmoiety.
 4. The method of claim 1, wherein the ActRIIa-Fc fusion proteincomprises the amino acid sequence of SEQ ID NO:2.
 5. The method of claim1, wherein the ActRIIa-Fc fusion protein is a dimer formed of twopolypeptides that each comprise an amino acid sequence that is at least95% identical to the amino acid sequence of SEQ ID NO:2, and wherein theActRIIa-Fc fusion protein comprises three or more sialic acid moieties.6. The method of claim 5, wherein the ActRIIa-Fc fusion proteincomprises the amino acid sequence of SEQ ID NO:2.
 7. The method of claim6, wherein the ActRIIa-Fc fusion protein comprises from three to fivesialic acid moieties.
 8. The method of claim 7, wherein the methodcauses less than a 10% increase in the patient's skeletal muscle mass.9. The method of claim 7, wherein the ActRIIa-Fc fusion protein isadministered so as to reach a serum concentration of the fusion proteinin the patient of at least 0.2 mg/kg.
 10. The method of claim 7, whereinthe ActRIIa-Fc fusion protein comprises an amino acid sequence that isat least 95% identical to SEQ ID NO:7.
 11. The method of claim 7,wherein the ActRIIa-Fc fusion protein has a serum half-life of between15 and 40 days in normal, healthy humans.
 12. The method of claim 7,wherein the ActRIIa-Fc fusion protein is administered to the patient nomore frequently than once per week.
 13. The method of claim 7, whereinthe ActRIIa-Fc fusion protein is administered to the patient no morefrequently than once per month.
 14. The method of claim 7, wherein theActRIIa-Fc fusion protein is administered to the patient no morefrequently than once per three months.
 15. The method of claim 1,further comprising administering a radiation therapy, endocrine therapyor cytotoxic agent to the human patient.
 16. The method of claim 1,wherein the human patient is a female with one or more risk factors forbreast cancer.
 17. The method of claim 1, wherein the ActRIIa-Fc fusionprotein comprises an amino acid sequence that is at least 95% identicalto SEQ ID NO:7.
 18. The method of claim 17, wherein the ActRIIa-Fcfusion protein comprises the amino acid sequence of SEQ ID NO:7.
 19. Themethod of claim 10, wherein the ActRIIa-Fc fusion protein comprises theamino acid sequence of SEQ ID NO:7.
 20. The method of claim 1, whereinthe patient is at risk for developing metastatic cancer.
 21. The methodof claim 1, wherein the patient has a primary breast cancer tumor orproliferative lesion of the breast, and wherein the ActRIIa-Fc fusionprotein is administered to the patient prior to the development ofmetastatic lesions.
 22. The method of claim 1, wherein the patient has aprimary breast cancer tumor or proliferative lesion of the breast, andwherein the ActRIIa-Fc fusion protein is administered to the patientprior to metastatic spread of cancer cells to the bone.
 23. The methodof claim 1, wherein the method is for treating estrogenreceptor-negative breast cancer in a human patient.
 24. The method ofclaim 23, wherein the ActRIIa-Fc fusion protein comprises a polypeptidecomprising the amino acid sequence of SEQ ID NO:2.
 25. The method ofclaim 23, wherein the ActRIIa-Fc fusion protein comprises a polypeptidecomprising an amino acid sequence that is at least 95% identical to SEQID NO:7.
 26. The method of claim 23, wherein the ActRIIa-Fc fusionprotein comprises a polypeptide comprising the amino acid sequence ofSEQ ID NO:7.
 27. The method of claim 23, wherein the ActRIIa-Fc fusionprotein is a dimer comprising two polypeptides that each comprise anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO:7.
 28. The method of claim 23, wherein theActRIIa-Fc fusion protein is a dimer comprising two polypeptides thateach comprise the amino acid sequence of SEQ ID NO:7.
 29. The method ofclaim 1, wherein the method is for preventing estrogen receptor-negativebreast cancer in a human patient.
 30. The method of claim 29, whereinthe ActRIIa-Fc fusion protein comprises a polypeptide comprising theamino acid sequence of SEQ ID NO:2.
 31. The method of claim 29, whereinthe ActRIIa-Fc fusion protein comprises a polypeptide comprising anamino acid sequence that is at least 95% identical to SEQ ID NO:7. 32.The method of claim 29, wherein the ActRIIa-Fc fusion protein comprisesa polypeptide comprising the amino acid sequence of SEQ ID NO:7.
 33. Themethod of claim 29, wherein the ActRIIa-Fc fusion protein is a dimercomprising two polypeptides that each comprise an amino acid sequencethat is at least 95% identical to the amino acid sequence of SEQ IDNO:7.
 34. The method of claim 29, wherein the ActRIIa-Fc fusion proteinis a dimer comprising two polypeptides that each comprise the amino acidsequence of SEQ ID NO:7.